登革2型病毒E基因DⅢ区的原核表达及血清学诊断应用

摘要

Objective To express the domain Ⅲ of Dengue virus type 2 envelope glycoprotein in Escherichia coli.and establish an in-house diagnostic assay for IgM antibodies against dengue viruses.Methods The DNA fragment for domain Ⅲ of the envelope glycoprotein was amplified by RT-PCR and cloned into vector pET-30a (+),so as to construct the expression plasmid pET30a-D2E-D Ⅲ.The recombinant plasmid was transformed into Escherichia coli competent cells BL2l (DE3) and induced by IPTG.The expressed products were analyzed by SDS-PAGE and Western blot.Purified by electroelution,the recombinant protein was attempted to establish an indirect ELISA assay for IgM antibody against Dengue viruses.in-house ELISA assay was assessed by conventional IFTA or Panbio Dengue IgM Capture ELISA.Results The 264 bp in length DNA fragment for domain Ⅲ was amplified and the expression plasmid pET30a-D2E-DⅢ was successfully constructed and expressed in E.coli BL2l(DE3).The indirect ELISA assay obtained a sensitivity of 93.75％ and a specificity of 91.25％ by comparing with IFTA.Furthermore,the in-house ELISA assay was consistent with results by using Panbio Dengue IgM Capture ELISA (x2 =0.214,P ＞ 0.05,Kappa =0.825).Conclusions The expression plasmid pET30a-D2E-D Ⅲ is stably expressed in E.coli BL2l (DE3) and the recombinant protein is suitalbe as a component antigen in serum diagnosis of dengue virus infection.%目的 表达登革2型病毒E基因DⅢ区重组蛋白并应用于血清登革病毒IgM抗体检测.方法 构建包含登革2型病毒E基因DⅢ区片段的重组表达质粒,并在E.coli BL21(DE3)中表达,利用重组蛋白建立间接ELISA法用于血清登革病毒IgM抗体检测.结果 成功构建重组表达质粒并表达重组蛋白,纯化的重组蛋白用于血清登革病毒IgM抗体检测,以IFTA作为参照标准,灵敏度为93.75％,特异度为91.25％;与Panbio Dengue IgM Capture ELISA试剂盒检测结果相比,二者具有较高的一致性(x2=0.214,P＞0.05;Kappa=0.825).结论 在大肠埃希菌中成功表达登革2型病毒E基因DⅢ区重组蛋白,并能够应用于血清登革病毒IgM抗体检测.